Association of IL-1B+3954 and IL-1RN Polymorphisms in Chronic Gastritis and Peptic Ulcer.

Background
Helicobacter pylori are the main cause of chronic inflammation and peptic ulcer. We aimed to determine if IL-1B+3954 and IL-1RN polymorphisms are associated with the risk of chronic gastritis and peptic ulcer in Iranian population.


Methods
In this case-control study, from 198 individuals enrolled by Mohammadi Hospital, Bandar Abbas, southern Iran from 2012 to 2014 and who showed the symptoms of chronic gastritis and 84 with peptic ulcer participated in the case group, two biopsies were taken from the body, antrum, or ulcer edge of each patient. Individuals without chronic gastritis or peptic ulcer were selected as the control group and we also confirmed the presence of anti-H. pylori serum IgG in 321 control subjects. IL-1B+3954C/T polymorphism was analyzed through PCR-RFLP, while the IL-1RN polymorphism was analyzed via PCR-based VNTR.


Results
IL-1B+3954 TT was associated with a high risk of gastritis and peptic ulcer [Odds Ratio (OR)]=2.63, 95% Confidence Interval (CI)= (1.47-4.70) (OR=3.40, CI=1.72-6.71) respectively and the IL-1B+3954 T allele was associated with chronic gastritis (OR=1.64, 95% CI=1.13-2.36). Moreover, patient carrying IL-1RN L/2 and allele 2 showed an increased risk of peptic ulcer (OR=2.97, CI=1.72-5.11, OR=1.64, CI=1.13-2.36), respectively.


Conclusion
IL-1B and IL-1RNare associated with an increased risk for chronic gastritis and peptic ulcer disease.


Introduction
Helicobacter pylori is a Gram-negative microaerophilic bacterium, whose infection is one of the major causes of chronic gastritis and peptic ulcer disease (PUD) in human beings (1,2). A majority of the world population is reported to be infected with H. pylori, but only 20% of the infected individuals develop clinically significant disease (3).
While most infected individuals remain asymptomatic, persistent colonization and chronic inflammation increase the risk of developing atrophic gastritis, peptic ulcers, and gastric cancer (4,5).The clinical outcome of H. pylori infection can be determined by the genetic characteristics of the host and the bacteria, and also by envi-ronmental factors (6).Recently, inflammatory response is one essential part of the pathogenesis of PUD. Evidence also suggests that inflammatory cytokines, besides the genetic variation in the genes that encode the inflammatory mediators, might play a key role in the development of PUD (7,8). The interleukin-1 (IL-1) gene cluster on chromosome 2q contains three related genes within a 430-kb region: IL-1A, IL-1B, and IL-1RN, which encode the pro-inflammation cytokines IL-1a and IL-1b and the endogenous receptor antagonist IL-1ra, respectively (9). IL-1b is up-regulated in the presence of H. pylori and is a potent inhibitor of gastric acid secretion (10). Three di-allelic polymorphisms in IL-1B have been reported, all representing C-T base transitions, at positions -511, -31, and +3954 bp. IL-1RN has a Penta-allelic 86-bp tandem repeat polymorphism in intron-2, while IL-1RN allele 2 has been shown to be associated with enhanced IL-1b production (11).Polymorphisms have been reported to be related to variations in the production levels of IL-1b and IL-1ra (12). In this study, we investigated whether IL-1B (rs1143634) and IL-1RN polymorphisms are associated with chronic gastritis or peptic ulcer in Iran.

Methods
We conducted a case-control study, 282 patients (including 198 patients with chronic gastritis and 84 patients with peptic ulcer) who underwent an endoscopic study in a specialized unit for Gastroenterology Endoscopy in the city of Bandar Abbas in 2012 to 2014, southern Iran were included in this study. All the study participants belonged to Hormozgan Province in Iran, and all of them suffered from functional dyspepsia and epigastric pain. These patients did not receive H. pylori eradication therapy. Moreover, we selected those individuals who had no signs of dyspepsia, no history of gastric diseases, and no indication for endoscopy, and used IgG-Anti H. pylori ELISA test to diagnose H. pylori infection in them. Finally, 321 people were positive for serum H. pylori IgG indicating their previous exposure to H. pylori. However, they had no signs of gastric diseases so they were selected as the control group. In both groups, the researcher excluded the subjects under non-steroidal anti-inflammatory drug (NSAID) treatment and those with autoimmune gastritis. Informed consent was obtained from the participants, and they were informed that the results would be used for developing better treatment strategies for such diseases. Hormozgan Bioethics Committee of the Medical University approved the validity of this study. Details of the study participants such as diet, socio-demographic factors, family history of gastritis or ulcers, and smoking habits were recorded in surveys.

Endoscopy and Gastric Histology
Every patient underwent an endoscopy using a video processor and a video gastroscope (Fujinon, Wayne, NJ, USA). In each patient, we took two biopsies from the antrum, corpus, or ulcer edge; one specimen was immediately fixed in formalin for histological testing and the other was placed in distilled water for the diagnostic test. The biopsies collected for the diagnostic test were stored at -20°C until the processing procedures. Furthermore, the histological sections were stained with hematoxylin and eosin and evaluated by a pathologist using updated Sydney System criteria (13). Endoscopic observation and histopathological confirmation were applied to determine the patient's pathology in the study. And we finally selected 282 individuals Helicobacter pylori positive as patient group.

Serology
Blood samples (5 ml) were collected from all the control group subjects. The serum was tested for IgG anti-H. pylori by ELISA; International Immuno-Diagnostics, Foster City, CA, USA) following the manufacturer's instructions. The sensitivity and specificity of this method was 96%. A subject was considered H. pylori positive if we detected antibodies in the serum.

Analysis of IL1B and IL1RN Genes
In this study, genomic DNA was extracted from peripheral blood using the QIA amp DNA Mini Kit. IL-1B+3954C/T polymorphisms were analyzed by RFLP with the TaqI enzyme (14). The PCR products were analyzed by electrophoresis on a 2% agarose gel stained with 0.1% ethidiumbromide. PCR products were digested with 2 units of TaqI for 8 h at 65°C and resulted in an intact fragment of 249 bp (allele T) or in two fragments of 135 and 114 bp (allele C).Fragments were analyzed by electrophoresis on 3% agarose gels stained with 0.1% ethidium bromide (Fig.1).

Statistical analysis
Hardy-Weinberg equilibrium (HWE) of allelic frequencies at individual loci was assessed by comparing the observed and expected genotype frequencies by incorporating the χ 2 test (for IL-1B P=0.197; for IL-1RN P=1.00). For statistical analysis, the χ 2 test was used to assess the statistical significance of the differences between the genotype and allelic frequencies of the population. Odds ratios (OR) with a 95% confidence interval (CI) were calculated for genotyping objectives. The level of significance was set at P<0.05.

Results
The case group represented 198 individuals with chronic gastritis and 84 patients with peptic ulcer in this study. The average age of the patients with gastritis was 41 yr with an age range of 17 to 85 yr; the peptic ulcer patients had an average age of 42 yr, ranging from 23 to 72 yr. Subjects in the control group had an average age of 41 yr and their age ranged from 21 to 74 yr. There were significant differences among the three groups in family history of ulcer and smoking habits ( Table 1). The relationships between individual IL-1B and IL-1RN genotypes among patients with a risk of chronic gastritis and peptic ulcer are represented in Tables 2 and 3.   There wassignificant association between the IL-1BTT genotype and peptic ulcer (OR=3.40, CI=1.72-6.71) but there was no significant association between TC genotype and T allele in peptic ulcer and control group. In peptic ulcer group there wassignificant association between L/2 genotype and 2 alleles as compared to control group (OR=2.97, CI=1.72-5.11and OR=1.64, CI=1.13-2.36). However, there were no differences in the frequency of the IL-1RN2/2 genotype between peptic ulcer and control.

Discussion
In this study, homozygous variant (TT) of IL-1B+3954 gene is associated with peptic ulcer and caring T allele (CT+TT) is correlated with gastritis. Heterozygous genotype (L/2) and caring 2 alleles of IL-1RN (L/2+ 2/2) an increased risk of peptic ulcer. Different pathological mechanisms related to the progression of chronic gastritis and peptic ulcer is suggested (12). H. pylori infection can cause hypochlorhydria, gastric atrophy, and eventually peptic ulcer and cancer. In fact, the interaction between the microorganism and the host immune system will lead to diseases. After H. pylori infection, the local immune cells are activated and exhibit immune responses affecting the disease process. For example, increased production of pro-inflammatory cytokines in the mucus (such as IL-1, IL-6, IL-10, TNF-α, and interferon-γ) of those who are at risk of H. pylori infection is associated with gastritis induced by H. pylori and the severity of inflammation (15). IL-1B is a member of the IL-1 family and a pro-inflammatory cytokine involved in the immune defense against infection. IL-1B expression increases the expression of factors adhered to vascular endothelial cells that cause the leukocytes to extravagate to the site of infection and resets the temperature regulation center in the hypothalamus which leads to increased body temperature. These are the activities of body immune system against infection. In addition, for the role of IL-1B in adjusting a secretion of acid and protecting cell activities in gastric mucus, there is a body of re-search related to IL-1B polymorphism, peptic and gastric ulcer. Also, it is reported that IL-1B is a cell-protecting molecule and strongly limits the gastric acid secretion (16). Therefore, IL-1 gene polymorphisms may be associated with the severity of inflammatory diseases, autoimmune diseases, such as rheumatoid arthritis, and cancers, including gastric cancer (17). In the context of such subjects, the effects of polymorphism in two cytokine genes, the proinflammatory cytokine IL-1B and antiinflammatory cytokine IL-1RN, in relation to the development of peptic ulcer and chronic gastritis were analyzed. In this study, a significant association was observed between IL-1B+3954TT and T allele genotype and patients with gastritis and TT genotype and peptic ulcer compared with the control group. Since the presence of TT polymorphism in IL-1B has been reported as associated with increased gene expression (18) and this increased expression enhances the severity of the gastric mucosal inflammatory responses (19), carriers of the TT genotype are susceptible to peptic ulcer and gastritis. Similar results have been obtained from the populations of Poland (20), African Americans and Caucasian (21), but this association was not found in the Chinese population (22). This difference in results can be due to different genetic backgrounds in different ethnic groups. In addition, we conducted on IL-1RN polymorphism, a significant association was also observed between the genotype L/2 and carriers of the allele 2 and peptic ulcer. IL-1RN is involved in gastric diseases. The allele 2 is associated with increased risk of precancerous lesions indicating the role of this polymorphism in the early stages of gastric carcinogenesis (23). In another study on the Caucasian population, the allele 2 had a significant association with increased risk of gastric cancer (24), but this association was not observed in the Korean population (11). In addition, in a study on the cytokine polymorphisms in which a population of Southern Brazil was involved, a significant association was observed between the frequency of the allele 2 and development of gastric cancer (25). These differences may be due to lower frequency of the risk allele in some populations, such as the Asian population. In this study, the frequencies of allele 2 and L/2were 46.4%, 42.9% and 25.2%, 19.6% in patients with peptic ulcer and control individuals, respectively. Since the allele 2 is associated with increased production of the IL-1B, which limits the amount of gastric acid and that acid reduction is favorable for bacterial colonization, the allele 2 can play a role in the development of gastric diseases with the presence of H. pylori (19).

Conclusion
This study supports the previous evidence on the role of inflammatory factors in development of gastric diseases in the Caucasian population which can potentially help to identify the patients at risk and to apply new methods of treatment in the future.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.